Needle array assembly and method for delivering therapeutic agents

ABSTRACT

A fluid delivery device includes an array of needles, each in fluid communication with a respective reservoir. Respective actuators are coupled so as to be operable to drive fluid from the reservoirs via needle ports. Each needle can have a plurality, of ports, and the ports can be arranged to deliver a substantially equal amount of fluid at any given location along its length. A driver is coupled to the actuators to selectively control the rate, volume, and direction of flow of fluid through the needles. The device can simultaneously deliver a plurality of fluid agents along respective axes in solid tissue in vivo. If thereafter resected, the tissue can be sectioned for evaluation of an effect of each agent on the tissue, and based on the evaluation, candidate agents selected or deselected for clinical trials or therapy, and subjects selected or deselected for clinical trials or therapeutic treatment.

CROSS REFERENCE

This application is a continuation application of U.S. application Ser.No. 15/449,714, filed on Mar. 3, 2017, which is a continuationapplication of U.S. application Ser. No. 15/214,392, filed on Jul. 19,2016, which is a continuation of U.S. application Ser. No. 13/489,594,filed Jun. 6, 2012, which is a continuation of U.S. application Ser. No.12/674,146, filed on Aug. 27, 2010, now U.S. Pat. No. 8,349,554, whichis the National Stage of International Application Serial No.PCT/US2008/073212, filed Aug. 14, 2008, which claims priority to theU.S. Provisional Application No. 60/955,676, filed on Aug. 14, 2007, allof which are incorporated herein by reference in their entirety.

BACKGROUND Technical Field

In general, the disclosed embodiments relate to devices and methods forthe introduction and subsequent evaluation of therapeutic agents tobiological tissue, and in particular to the simultaneous introduction ofa plurality of agents to the tissue in viva

Description of the Related Art

Numerous cancer-related therapeutics are under phase I or phase IIclinical trial and evaluations at any particular time; however, most ofthem will fail to advance. In fact, it is estimated that more than 90%of cancer-related therapeutics will fail phase I or II clinical trialevaluation. The failure rate in phase III trials is almost 50%, and thecost of new drug development from discovery through phase III trials isbetween $0.8 billion and $1.7 billion and can take between eight and tenyears.

In addition, many patients fail to respond even to standard drugs thathave been shown to be efficacious. For reasons that are not currentlywell understood or easily evaluated, individual patients may not respondto standard drug therapy. One significant challenge in the field ofoncology is to exclude drug selection for individual patients havingcell autonomous resistance to a candidate drug to reduce the risk ofunnecessary side effects. A related problem is that excessive systemicconcentrations are required for many oncology drug candidates in effortsto achieve a desired concentration at a tumor site, an issue compoundedby poor drug penetration in many under-vascularized tumors (Tunggal etal., 1999 Clin. Canc. Res. 5:1583).

Clearly there is a need in the art for improved devices and methods fortesting and delivering cancer therapies, including improvedmethodologies for performing efficient pre-clinical and clinical studiesof candidate oncology medicines, and for identifying therapeutics havingincreased likelihood of benefitting individual subjects. The presentinvention addresses these and similar needs, and offers other relatedadvantages.

BRIEF SUMMARY

It is an aspect of the present invention to provide a device fordelivery of a fluid to a solid tissue, comprising: a plurality ofneedles arranged in an array; a plurality of reservoirs, each in fluidcommunication with a respective one of the plurality of needles; and aplurality of actuators operatively coupled to respective ones of theplurality of reservoirs and configured to control a fluid pressurewithin the reservoir. In certain embodiments each of the plurality ofactuators comprises one of a plurality of plungers, a first end of eachof the plurality of plungers being received in a respective one of theplurality of reservoirs, and in certain further embodiments the plungersof the plurality of plungers are operatively coupled together atrespective second ends so as to be simultaneously depressable. Certainstill further embodiments comprise a plunger driver configured todepress all of the plurality of plungers at a selectively variable rate.In other embodiments each of the plurality of actuators comprises one ofa plurality of fluid transmission lines having first and second ends, afirst end of each of the plurality of fluid transmission lines beingcoupled to a respective one of the plurality of reservoirs. In otherembodiments the device comprises a fluid pressure source, and each ofthe plurality of actuators comprises a fluid coupling between the fluidpressure source and a respective one of the plurality of reservoirs. Infurther embodiments the fluid pressure source comprises at least one ofa compressor, a vacuum accumulator, a peristaltic pump, a mastercylinder, a microfluidic pump, and a valve. In another embodiment, eachof the plurality of needles comprises a plurality of ports distributedalong its length.

In another embodiment there is provided a device for delivering a fluidto a solid tissue, comprising a dispenser including a needle having aplurality of ports distributed along a length thereof, a reservoir influid communication with the dispensing needle, and a plunger having afirst end positioned in the reservoir; and a plunger driver coupled to asecond end of the plunger and configured to depress the plunger at aselectably variable rate. In certain further embodiments the dispenseris one of a plurality of dispensers arranged in a dispenser array, eachcomprising a needle, a reservoir, and a plunger having first and secondends. In certain further embodiments the plunger driver is coupled tothe second end of the plunger of each of the plurality of dispensers andis configured to depress each of the plungers simultaneously. In certainother further embodiments the device comprises a plurality ofcylindrical tubes arranged in an array corresponding to the dispenserarray, each of the plurality of cylindrical tubes being sized andpositioned to receive the needle of a respective one of the plurality ofdispensers.

In certain other embodiments the plunger driver comprises a driver shaftcoupled to the plunger and having a threaded region, the plunger driverconfigured such that rotation of the driver shaft in a first directiondepresses the plunger a distance corresponding to a thread pitch of thethreaded region and a number of revolutions of the driver shaft. Incertain further embodiments the device comprises a motor having a rotorcoupled to the driver shaft of the plunger driver such that the rotorand the driver shaft are rotationally fixed with respect to each other,the motor being controllable to rotate the rotor at a selectablyvariable rate. In certain other further embodiments the device comprisesa motor having a rotor coupled to the driver shaft of the plunger driversuch that the rotor and the driver shaft are rotationally fixed withrespect to each other, the motor being controllable to rotate the rotorto a selectable angle of rotation. Certain further embodiments comprisea controller coupled to the motor, the controller being programmable tocontrol direction and speed of rotation of the rotor and to control anumber of degrees from a start of rotation to an end of rotation. Inother embodiments of the above described device, the dispenser comprisesa dispenser cylinder; a first portion of the dispenser cylinder definesthe reservoir; and a second portion of the dispenser cylinder definesthe needle. In another embodiment the plurality of ports are sized andpositioned along the length of the needle so as to deliver asubstantially equal amount of fluid at any given location along thelength of the needle. In another embodiment the plurality of ports isevenly distributed along a portion of the length of the needle.

In certain embodiments a size of each of the plurality of ports isinversely related to a distance of the respective port from a tip-end ofthe needle. In certain other embodiments a distribution density of theplurality of ports is inversely related to a distance of the respectiveport from a tip-end of the needle. In certain other embodiments theplurality of ports is distributed in a spiral pattern along the lengthof the needle. In certain other embodiments the plurality of ports isarranged in pairs of ports on opposite sides of the needle, with eachpair of ports rotated 90 degrees with respect to adjacent pairs of portsalong the length of the needle.

According to certain other embodiments disclosed herein, there isprovided a method, comprising placing an agent in a reservoir of each ofa plurality of dispenser needles; inserting each of the plurality ofdispenser needles into a selected region of solid tissue; andintroducing the agent in the reservoirs into the selected region ofsolid tissue by simultaneously overpressurizing each of the plurality ofdispenser needles. In certain further embodiments the introducingcomprises introducing the agent in the reservoirs into the selectedregion of solid tissue from a plurality of apertures along each of theplurality of dispenser needles. Certain other further embodimentscomprise at least one of imaging the solid tissue prior to theinserting, imaging the solid tissue concurrently with the inserting, andimaging the solid tissue after the inserting. In certain other furtherembodiments the inserting comprises inserting an array of introducerneedles into a subject; inserting each of the plurality of dispenserneedles into a respective one of the array of introducer needles; andextending a tip-end of each of the plurality of dispenser needles beyonda tip end of the respective one of the array of introducer needles andinto the selected region of tissue. Certain further embodiments compriseremoving stylets from the introducer needles of the array prior toinserting the plurality of dispenser needles.

In certain embodiments the selected region of tissue is a portion of atumor in a subject, and in certain further embodiments the subject isone of a preclinical model and a human patient. In certain otherembodiments the method comprises excising at least the portion of thetumor after the introducing. Certain further embodiments comprise atleast one of imaging the tumor prior to the excising, imaging the tumorconcurrently with the excising, and imaging the tumor after to theexcising. In certain other embodiments the excising comprises excisingat least the portion of the tumor at a time that is a selected period oftime after introducing the agent. In certain further embodiments theselected period of time is one of a range of time, a minimum period oftime for excising, and a specific period of time for excising. Incertain embodiments the selected period of time is a period exceeding 48hours. In certain embodiments the selected period of time is a range ofbetween about 72 and about 96 hours. In certain embodiments the selectedperiod of time is a period exceeding one week.

According to certain other embodiments of the above described method,the agent comprises a plurality of agents, and the placing comprisesplacing each of the plurality of agents into the reservoir of arespective one of the plurality of dispenser needles. In certain furtherembodiments the plurality of agents comprises at least one of a negativecontrol composition and a positive control composition. In certain otherfurther embodiments the plurality of agents comprises at least oneposition marker. In certain other further embodiments at least one ofthe plurality of agents is a candidate effective agent. In certain otherfurther embodiments at least one of the plurality of agents comprises anindicator of efficacy, which in certain further embodiments comprises atleast one of a nanoparticle, a nanostucture, and an indicator dye. Incertain other embodiments at least one of the plurality of agents isselected based on a clinically demonstrated efficacy of the respectiveagent. In certain other further embodiments of the above describedmethod, the method comprises assessing, with respect to at least one ofthe plurality of agents, at least one of efficacy, activity, andtoxicity of the agent.

In another embodiment there is provided a method for identifyingrelative efficacies of a plurality of agents for treating a subject,comprising injecting each of a plurality of candidate effective agentsinto a respective location in an injection site in a solid tissue in asubject; excising from the subject at least the injection site of thesolid tissue; and evaluating the excised injection site for an alteredphysiologic state at each of the respective locations, and therefromidentifying relative efficacies of the plurality of agents. In certainfurther embodiments the excising comprises one of excising at least 48hours after the injecting, excising at least 72 hours after theinjecting, excising 72 to 96 hours after the injecting, and excising atleast one week after the injecting.

In another embodiment there is provided a method of operation of atherapeutic device, comprising charging a reservoir of each of aplurality of needles with a respective one of a plurality of agents;injecting, simultaneously, each of the plurality of agents into arespective region of a solid tissue; and evaluating an effect of each ofthe plurality of agents on the respective region. In certain furtherembodiments the injecting comprises injecting the plurality of agentsinto the solid tissue in vivo, and in certain still further embodimentsthe method comprises excising the solid tissue prior to the evaluating.In certain embodiments the method comprises imaging the solid tissue,which in certain further embodiments comprises imaging the solid tissuein vivo. In certain other embodiments the injecting comprisesdistributing each of the plurality of agents into the solid tissue alongan axis in the respective region of the tissue. In certain otherembodiments the method further comprises assessing, with respect to atleast one of the plurality of agents, at least one of efficacy,activity, and toxicity of the agent.

Also provided herein according to certain embodiments is a method ofdetermining efficacy of a cancer treatment regimen, comprisingsimultaneously introducing an agent to a plurality of positions in asolid tumor in a subject in vivo; removing the tumor from the subject;and evaluating an effect of the agent on the tumor in vitro. In certainfurther embodiments the agent comprises a plurality of agents and theintroducing comprises distributing each of the plurality of agents to arespective one of the plurality of positions in the tumor. In anotherembodiment there is provided a method, comprising introducing an agentto a region of solid tissue in a subject by distributing the agent to aplurality of positions along an axis within the region of solid tissuein vivo; removing the region of solid tissue from the subject; andevaluating an effect of the agent on the region of solid tissue invitro. In a further embodiment the region of solid tissue comprises atumor.

In certain embodiments the axis is one of a plurality of parallel axesin the region of solid tissue, and wherein the introducing comprisesdistributing the agent along each of the plurality of parallel axes. Incertain further embodiments the introducing comprises simultaneouslydistributing the agent along each of the plurality of parallel axes, andin certain other further embodiments the plurality of parallel axes isarranged in an array. In certain other embodiments the method comprisesintroducing at least two position markers to the region of solid tissuealong a respective one of the plurality of parallel axes, and in certainfurther embodiments the introducing at least two position markerscomprises distributing the at least two position markers alongrespective parallel axes within the region of solid tissue. In certainother embodiments the at least two position markers each comprise adetectable label that is selected from the group consisting of aradiolabel, a radio-opaque label, a fluorescent label, a colorimetriclabel, a dye, an enzymatic label, a GCMS tag, avidin, and biotin.

In certain other embodiments of the above described method, the agent isone of a plurality of agents and the axis is one of a plurality ofparallel axes arranged in an array in the region of solid tissue, andwherein the introducing comprises distributing each of the plurality ofagents to a plurality of positions along a respective one of theplurality of parallel axes. In certain other embodiment the methodcomprises at least one of imaging the solid tissue prior to theintroducing, imaging the solid tissue concurrently with the introducing,and imaging the solid tissue after the introducing. In certain otherembodiments the evaluating comprises sectioning the region of solidtissue into a plurality of sections normal to the parallel axes. Incertain further embodiments the evaluating comprises detecting withinthe solid tissue an altered physiologic state that results from at leastone of the plurality of agents. In certain further embodiments thedetecting comprises, with respect to the at least one of the pluralityof agents, at least one of detecting a degree of permeation of the agentthrough the solid tissue, detecting a physicochemical effect of theagent on the tissue, and detecting a pharmacological effect of the agenton the tissue. In certain other embodiments the evaluating comprisesdetermining the effects of at least two of the plurality of agents on asame position within the region of the solid tissue. In certain otherembodiments the evaluating comprises determining the effects of at leasttwo of the plurality of agents on adjacent positions within the regionof the solid tissue.

In certain other embodiments the evaluating comprises differentiating adegree of the effect of at least one of the plurality of agents ondifferent sections of the solid tissue according to differentcharacteristics of the different sections of the solid tissue. Incertain other embodiments the evaluating comprises comparing a firsteffect of at least a first one of the plurality of agents on the solidtissue with a second effect of at least a second one of the plurality ofagents on the solid tissue. In certain other embodiments the evaluatingcomprises, with respect to at least one of the plurality of agents,assessing at least one of efficacy, activity, and toxicity on the regionof solid tissue. In certain other embodiments the method comprisesdeselecting at least one of the plurality of agents based on theevaluating. In certain other embodiments the method comprises selectingat least one of the plurality of agents based on the evaluating. Incertain other embodiments the method comprises prioritizing at least twoof the plurality of agents based on the evaluating. In certain otherembodiments the method comprises distributing the plurality of agents toa plurality of positions, each along a respective one of a plurality ofparallel axes within a region of solid tissue within each of a pluralityof subjects. In certain further embodiments the method comprises one of(i) selecting at least one of the plurality of agents based on theevaluating, (ii) deselecting at least one of the plurality of agentsbased on the evaluating, and (iii) prioritizing at least two of theplurality of agents based on the evaluating. In certain otherembodiments the method comprises one of (i) selecting at least one ofthe plurality of subjects based on the evaluating, (ii) deselecting atleast one of the plurality of subjects based on the evaluating, and(iii) prioritizing at least two of the plurality of subjects based onthe evaluating. In certain other embodiments the evaluating comprisesdetermining a level of altered physiologic state of the solid tissuenear at least one of the plurality of parallel axes.

Turning to another embodiment there is provided a fluid agent-deliveringdevice comprising (i) a plurality of needles arranged in an array, eachof said needles having, independently, one or a plurality of portsdistributed along its length wherein at least one needle has saidplurality of ports, (ii) a plurality of reservoirs containing the fluidagent, each of said reservoirs being in fluid communication with arespective one of the plurality of needles, and (iii) a plurality ofplungers, a first end of each plunger being received in a respective oneof the plurality of reservoirs and a second end of each plunger beingdepressable such that depressing each plunger results in injection ofthe fluid agent through the respective one of the plurality of needles.

In another embodiment of the presently disclosed invention there isprovided a method for selective delivery of a fluid agent to a solidtissue, comprising (a) introducing a plurality of needles of a fluidagent-delivering device into the solid tissue; and (b) administering thefluid agent into the solid tissue by injection through said needles. Incertain further embodiments the solid tissue has been removed from asubject. In certain other further embodiments the solid tissue is in asubject. In certain further embodiments the agent is delivered to thesolid tissue in a therapeutically effective amount. In certain stillfurther embodiments, outside the solid tissue, the agent is either (i)undetectable, or (ii) if detectable outside the solid tissue, the agentis present at less than a minimal dose. In certain embodiments the solidtissue comprises a tumor. In certain further embodiments the tumor isselected from a benign tumor and a malignant tumor. In certain otherfurther embodiments the tumor is selected from a primary tumor, aninvasive tumor and a metastatic tumor. In certain other furtherembodiments the tumor comprises at least one cancer cell selected from aprostate cancer cell, a breast cancer cell, a colon cancer cell, a lungcancer cell, a brain cancer cell, and an ovarian cancer cell. In certainother further embodiments the tumor comprises a cancer selected fromadenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma,small cell carcinoma, large cell undifferentiated carcinoma,chondrosarcoma and fibrosarcoma. In certain other embodiments the solidtissue is selected from brain, liver, lung, kidney, prostate, ovary,spleen, lymph node, thyroid, pancreas, heart, skeletal muscle,intestine, larynx, esophagus and stomach.

In certain other embodiments the fluid agent comprises an agent that isselected from (a) a gene therapy agent; (b) a chemotherapy agent; (c) asmall molecule; (d) an antibody; (e) a protein; (f) one of a smallinterfering RNA and an encoding polynucleotide therefor; (g) one of anantisense RNA and an encoding polynucleotide therefor; (h) one of aribozyme and an encoding polynucleotide therefor; (i) a detectablelabel; and (j) one of a therapeutic protein, polypeptide, and apeptidomimetic. In certain further embodiments the detectable label isselected from a radiolabel, a radio-opaque label, a fluorescent label, acolorimetric label, a dye, an enzymatic label, a GCMS tag, avidin, andbiotin. In certain embodiments the agent is selected from (i) a genetherapy agent that comprises at least one operably linked promoter, (ii)a small interfering RNA-encoding polynucleotide that comprises at leastone operably linked promoter; (iii) an antisense RNA-encodingpolynucleotide that comprises at least one operably linked promoter; and(iv) a ribozyme-encoding polynucleotide that comprises at least oneoperably linked promoter. In certain further embodiments the operablylinked promoter is selected from a constitutive promoter and aregulatable promoter. In certain still further embodiments theregulatable promoter is selected from an inducible promoter, a tightlyregulated promoter and a tissue-specific promoter.

In certain other embodiments there is provided a method for altering aphysiologic state in a solid tissue, comprising: (a) introducing aplurality of needles of a fluid agent-delivering device into the solidtissue; and (b) administering the fluid agent into the solid tissue byinjection through said needles.

In certain embodiments there is provided a method for obtainingbiological samples from a plurality of positions in a solid tissue,comprising (a) introducing a multiple needle device into the solidtissue, thereby placing a plurality of needles at a plurality ofpositions in the tissue; and (b) generating negative pressure at a portof each needle of said multiple needle device under conditions and for atime sufficient to draw into said needles a plurality of biologicalsamples from said plurality of positions in the tissue, and therebyobtaining biological samples from a plurality of positions in thetissue.

In certain embodiments there is provided a method for obtainingbiological samples from a plurality of positions along an axis in asolid tissue, comprising (a) introducing a multiple needle device intothe solid tissue, thereby placing a plurality of needles at a pluralityof positions in the tissue; and (b) generating negative pressure at aplurality of ports located along a length of each needle of saidmultiple needle device under conditions and for a time sufficient todraw into said needles a plurality of biological samples from saidplurality of positions in the tissue, and thereby obtaining biologicalsamples from a plurality of positions along an axis in the tissue.

In certain embodiments there is provided a method of screening subjectsfor eligibility to participate in a clinical trial of one or moreagents, comprising (a) introducing one or more agents to a region ofsolid tissue in one or more subjects in vivo by distributing each ofsaid agents to a plurality of positions along an axis within the regionin each subject; (b) removing the region of solid tissue from each ofsaid subjects; and (c) evaluating each region removed in (b) for aneffect of each agent on the respective position along the axis withinthe region, wherein either (i) for any given agent or agents presence ofa detectable effect of said agent or agents on the solid tissue regionfrom the subject indicates eligibility of the subject for participationin a clinical trial of the agent or agents, (ii) for any given agent oragents absence of a detectable effect of said agent or agents on thesolid tissue region from the subject indicates ineligibility of thesubject for participation in a clinical trial of the agent or agents, or(iii) both (i) and (ii).

In certain embodiments there is provided a method of rating a candidateagent for development into a therapeutic agent for treating a solidtumor, comprising (a) introducing one or more candidate agents to aregion of a solid tumor of known tumor type in each one or more subjectshaving a tumor of the known tumor type, by distributing each of saidcandidate agents to a plurality of positions along an axis within theregion in each subject; (b) removing the region of solid tumor from eachof said subjects; and (c) comparing each region removed in (b) for aneffect of each candidate agent on the respective position along the axiswithin the region, wherein an agent that results in a greater beneficialeffect when introduced to the tumor receives a higher rating fordevelopment into a therapeutic agent for treating the solid tumor, andan agent that results in a lesser beneficial effect when introduced tothe tumor receives a lower rating for development into a therapeuticagent for treating the solid tumor.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic diagram of a needle array assembly for injectingbiological tissue with therapeutic agents according to variousembodiments.

FIGS. 2A-2D and 3 show delivery needles according to respectiveembodiments.

FIGS. 4A and 4B show portions of a delivery needle and an insertionneedle in, respectively, an insertion position and a delivery position.

FIG. 5 is a diagrammatic view of a delivery assembly according to anembodiment.

FIG. 6 shows a portion of a needle array, including a reservoir,according to an embodiment.

FIG. 7 shows elements of a delivery assembly according to anotherembodiment.

FIG. 8 is a diagrammatic view of a delivery assembly according to afurther embodiment.

FIG. 9 shows diagrammatically a portion of a tumor illustratingprinciples of the invention.

FIG. 10 is a diagram of a data processing system according to anembodiment.

DETAILED DESCRIPTION

The present invention is directed in certain embodiments as describedherein to devices and methods for delivery of fluids to solid tissues,and in particular embodiments, to solid tumors. The herein describedembodiments relate in part to certain surprising and heretoforeunrecognized advantages, disclosed in greater detail below, that derivefrom exquisite control of the location, amount and time of fluiddelivery to solid tissue. These and related embodiments feature theprecise positioning of delivery needle outlet apertures, includingpositioning of spatially defined multiple-needle arrays and/or ofneedles having multiple outlet apertures at defined locations, andfurther including the use of fluidics configurations that provideextremely fine control over fluid delivery events. The inventionprovides improved accuracy and versatility to screening therapeuticcompounds such as anti-cancer agents for use in treating solid tumors,and permits early exclusion from a screening program or a therapeuticregimen of candidate drugs to which tumor cells may be resistant.

Accordingly, for example, certain embodiments contemplate direct drugdelivery to a solid tissue at low flow rates with low shear forces thateliminate or reduce mechanochemical damage to tissues while permittingprecisely targeted therapeutic agent delivery to defined focal sites.These and related embodiments permit advantageous and selective deliveryof a therapeutic agent to a solid tissue in vivo in a therapeuticallyeffective amount, while in further related embodiments the agent isundetectable outside the solid tissue or is present at less than aminimal dose. Hence, problems (e.g., toxicity, detrimental side-effects,etc.) associated with administering excessively high systemicconcentrations in order to obtain a therapeutically effectiveconcentration in a desired solid tissue are overcome by the presentlydisclosed embodiments.

Additionally, certain embodiments contemplate direct delivery ofmultiple drugs, candidate drugs, imaging agents, positional markers,indicators of efficacy and appropriate control compositions to aplurality of spatially defined locations along parallel axes in a solidtissue, such as a solid tumor, followed, after a desired time interval,by excision of the treated tissue and evaluation or analysis of thetissue for effects of the treatments. Indicators of efficacy may be, forexample, detectable indicator compounds, nanoparticles, nanostructuresor other compositions that comprise a reporter molecule which provides adetectable signal indicating the physiological status of a cell, such asa vital dye (e.g., Trypan blue), a colorimetric pH indicator, afluorescent compound that may exhibit distinct fluorescence as afunction of any of a number of cellular physiological parameters (e.g.,pH, intracellular Ca²⁺ or other physiologically relevant ionconcentration, mitochondrial membrane potential, plasma membranepotential, etc., see Haugland, The Handbook: A Guide to FluorescentProbes and Labeling Technologies (10^(th) Ed.) 2005, Invitrogen Corp.,Carlsbad, Calif.), an enzyme substrate, a specific oligonucleotideprobe, a reporter gene, or the like. Control compositions may be, forexample, negative controls that have been previously demonstrated tocause no statistically significant alteration of physiological state,such as sham injection, saline, DMSO or other vehicle or buffer control,inactive enantiomers, scrambled peptides or nucleotides, etc.; andpositive controls that have been previously demonstrated to cause astatistically significant alteration of physiological state, such as anFDA-approved therapeutic compound.

Typically and in certain preferred embodiments, the excised tissue maybe cut into a plurality of serial histological sections along parallelplanes that are substantially normal (e.g., perpendicular or deviatingfrom perpendicular by as much as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 20, 25, 30, 35 or more degrees) to the parallel axes, foranalysis by any of a number of known histological, histochemical,immunohistological, histopathologic, microscopic (including morphometricanalysis and/or three-dimensional reconstruction), cytological,biochemical, pharmacological, molecular biological, immunochemical,imaging or other analytical techniques, which techniques are known topersons skilled in the relevant art. See, e.g., Bancroft and Gamble,Theory and Practice of Histological Techniques (6^(th) Ed.), 2007Churchill Livingstone, Oxford, UK; Kiernan, Histological andHistochemical Methods: Theory and Practice, 2001 Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; M. A. Hayat (Ed.), CancerImaging—Vols. 1 and 2, 2007 Academic Press, NY. Imaging may be performedbefore, during or after dispenser needles are inserted into the solidtissue. Positional markers are known and include, as non-limitingexamples, metal or plastic clips, fluorescent quantum dots, India ink,metal or plastic beads, dyes, stains, tumor paint (Veiseh et al., 2007Canc. Res. 67:6882) or other positional markers, and may be introducedat desired positions. Markers may include any subsequently locatablesource of a detectable signal, which may be a visible, optical,colorimetric, dye, enzymatic, GCMS tag, avidin, biotin, radiological(including radioactive radiolabel and radio-opaque), fluorescent orother detectable signal.

A detectable marker thus may comprises a unique and readily identifiablegas chromatography/mass spectrometry (GCMS) tag molecule. Numerous suchGCMS tag molecules are known to the art and may be selected for usealone or in combination as detectable identifier moieties. By way ofillustration and not limitation, various different combinations of one,two or more such GCMS tags may be added to individual reservoirs of thedevice described herein in a manner that permits the contents of eachreservoir to be identified on the basis of a unique GCMS “signature”,thereby permitting any sample that is subsequently recovered from aninjection region to be traced back to its needle of origin foridentification purposes. Examples of GCMS tags includeα,α,α-trifluorotoluene, α-methylstyrene, o-anisidine, any of a number ofdistinct cocaine analogues or other GCMS tag compounds having readilyidentifiable GCMS signatures under defined conditions, for instance, asare available from SPEX CertiPrep Inc. (Metuchen, N.J.) or fromSigmaAldrich (St. Louis, Mo.), including Supelco® products described inthe Supelco® 2005 gas chromatography catalog and available fromSigmaAldrich.

Through the use of the device described herein, which includesconfiguration (e.g., by placing at least one positional marker in one ormore known locations) of the multiple needles in a manner that permitsready identification of the effects at a particular location, if any, ofthe contents released from a particular needle at the tissue locationthese and related embodiments thus contemplate methods of simultaneouslycomparing the relative therapeutic efficacies and/or toxicities of alarge number of candidate therapeutic agents. Such applications may finduses in methods of drug screening and drug discovery, such as inpreclinical animal models to identify and functionally characterizepotential new therapeutics. For instance, a plurality of siRNAs may beadministered intratumorally and their relative abilities to knock downexpression of a desired target gene may be compared. Other similarembodiments may find uses in clinical contexts, for example, to“deselect”, or eliminate from consideration, known therapeutic agentsthat have no effect in a particular tumor, thereby advantageouslyadvancing the therapeutic management of a patient by avoiding the lossof time and the undesirable side-effects that may be associated withadministering an ineffectual treatment regimen.

The present invention provides compositions and methods that are usefulfor the classification and/or stratification of a subject or patientpopulation, including for use in drug discovery and in pharmacogenomics.In these and related embodiments, correlation of one or more indicia ofan altered physiological state with a position at which a givencandidate agent has been introduced in a solid tumor may be used togauge the subject's responsiveness to, or the potential efficacy of, aparticular therapeutic treatment; related embodiments contemplate thisapproach for “deselection”, or elimination from consideration aspotential therapies, of candidate agents in which no evidence of analtered physiological state is detected at a site of introducing in thetumor.

As described herein, determination of levels of at least one indicatorof altered physiologic state may also be used to stratify a patientpopulation for eligibility to participate in a clinical trial. These andrelated embodiments are contemplated as usefully providing advantagesassociated with evaluation of candidate therapeutic compounds at anearlier stage of development than is currently the case. For instance,it is not currently standard clinical trial practice to establishbiomarker parameters (which may be the basis for exclusion of subjects)prior to Phase III studies, whereas the embodiments described herein mayprovide useful results even in the absence of established biomarkercriteria, for example, at Phase II. Accordingly it is envisioned thatthrough the practice of certain presently disclosed embodiments,relevant information on the properties of a candidate agent may beobtained earlier in a solid tumor oncology drug development program thanhas previously been the case, including in a manner which maytime-efficiently and cost-effectively permit elimination from a clinicaltrial of subjects for whom no response or benefit can be expected basedon a nonresponder result for a particular candidate agent.

For example, stratification of a patient population according to levelsof at least one indicator of altered physiologic state, determined asdescribed herein, may provide a useful marker with which to correlatethe efficacy of any candidate therapeutic agent being used in cancersubjects, and/or to classify subjects as responders, nonresponders orpossible responders.

Referring first to FIG. 1, a needle array assembly 100 is shown,including a plurality of needles 112, a plurality of reservoirs 114, aplurality of delivery actuators such as, in the present example,plungers 116, and a controller 102. Each of the plurality of needles 112is fixed in position relative to the others of the plurality of needles,and the plungers are likewise operatively coupled so as to be fixed inposition and simultaneously actuable. Each of the plurality of needles112 is in fluid communication with a respective one of the plurality ofreservoirs 114, and each of the plurality of plungers includes a firstend positioned in a respective one of the plurality of reservoirs 114.The controller 102 is operatively coupled to second ends of each of theplurality of plungers 116. The controller is configured to controlactuation of the plungers within the reservoir with respect to speed,distance, and direction of movement.

Movement of the plurality of plungers 116 in a first direction creates anegative pressure in the respective reservoirs 114, drawing atherapeutic agent or other fluid into the reservoirs via the respectiveneedle 112, thereby charging the reservoirs. Each reservoir 114 can becharged with a different agent, or some or all of the reservoirs can becharged with a common agent. Movement of the plurality of plungers 116in a second direction creates a positive pressure, or overpressure, inthe respective reservoirs 114, forcing the contents of the reservoirsout via the respective needles 112.

In this configuration, a relatively small amount of a plurality oftherapeutic agents can be simultaneously delivered directly to a regionof solid tissue 106 for evaluation and analysis. In some embodiments,the amount of a therapeutic agent delivered to the tissue is less than 1μL per needle. The evaluation of the tissue 106 and the efficacy of thedifferent therapeutic agents delivered thereto can be used, for example,to screen potential therapeutic agents for subsequent clinical trials orto make patient-specific treatment decisions based on the relativeefficacy of the therapeutic agents in the tissue 106.

According to various embodiments, any number of needles can be used. Forexample, as few as one, two, or three needles can be used, and accordingto some embodiments, more than one thousand needles can be used.According to an embodiment, each of the needles includes a plurality ofports or apertures arranged along the length of the needle.

Turning now to FIGS. 2A-2D, various configurations of needles 120 areshown. FIG. 2A shows a delivery needle 120 a including a plurality ofports 122 in pairs on opposite sides of the needle, the pairs beingevenly spaced along its length. Each pair is rotated 90 degrees withrespect to adjacent pairs of ports along the length of the needle 120 a.When fluid in a reservoir in fluid communication with the needle 120 ais subjected to an overpressure, it is forced from the needle via theplurality of apertures 122. Because the reservoir holding the fluid isto the right of the needle 120 a, as viewed in the figures, anoverpressure in the reservoir will result in the largest volume of fluidbeing forced from the right-most ports 122, such that the fluid will bedelivered in a progressively diminishing volume along its length towardthe tip-end 124. The relative volume of fluid distributed from each ofthe plurality of ports 122 along the needle 120 a may be influenced by anumber of factors including, for example, viscosity of the fluid, thesize and concentration of solids suspended therein, the density,permeability, and wettability of tissue in which the needle ispositioned, the degree of overpressure, the size of the ports, etc.

FIG. 2B shows a delivery needle 120 b according to another embodiment,in which ports 122 are largest near the tip-end 124 of the needle 120 b,and the relative size of each of the plurality of ports is inverselyrelated to a distance of the respective port from the tip-end of theneedle. Thus, while an overpressure of fluid in the needle will begreatest at the right-most port 122, that will also be the smallestport, and, conversely, while the overpressure will be lowest at theleft-most port 122, that port will also be the largest. By appropriatesizing of each of the ports 122, the needle 120 b can be configured todeliver a substantially equal volume of fluid at any given locationalong its axis, or alternatively, the needle can be configured todeliver fluid according to any selected distribution profile along itsaxis, by appropriate selection of the size of the respective ports 122.The size of the apertures can vary along the length of the needle fromabout 0.01 mm or less to about 0.25 mm or more.

FIG. 2C shows a delivery needle 120 c according to an embodiment inwhich a distribution density of the plurality of ports 122 is inverselyrelated to a distance of the respective port from the tip-end 124 of theneedle 120 c. In other words, the ports 122 closest to the tip-end 124of the needle 120 c are the most closely spaced, while the spacingbetween the ports grows increasingly greater as the distance from thetip-end increases. Accordingly, when fluid in the associated reservoiris subjected to an overpressure, the volume of fluid per port 122 willbe greatest at the right-most port, but a lower volume of fluid per portwill be offset toward the left by the progressively closer spacing ofthe ports. Thus, the overall distribution of fluid along the length ofthe needle 120 c can be made to be substantially consistent bydistributing the ports 122 as described above, or can be made to conformto another selected distribution profile by appropriate selection of thedistribution density of the ports along the needle.

Turning to FIG. 2D, a delivery needle 120 d is shown according toanother embodiment. Ports 126 of the needle 120 d are formed in a spiralpattern, with each port rotated 90° with respect to adjacent ports. Inthe embodiment shown in FIG. 2D, the ports 126 are formed bywire-electrode electrical discharge machining (wire EDM). In cutting theports 126, the depth and the length of each cut can be selected tocontrol the port size, while the pitch of the spiral can be selected tocontrol the distribution density. Thus, ports 126 configured as shown inFIG. 2D can be differentially sized or spaced as described withreference to FIGS. 2B and 2C.

In addition to wire EDM, the ports 122, 126 of the needles 120 can beformed by any appropriate method, including, for example, laser cutting,waterjet cutting, chemical etching, mechanical drilling or grinding,etc.

The tip-ends 124 of the needles 120 are shown as being closed andpointed. According to some research, “pencil point” needles, such as,for example, Sprotte and Whitacre needles, may be less damaging tobiological tissue than bevel-tipped needles. Additionally, fluidsinjected into tissue using pencil point side-port needles tend to remainin the tissue rather than leaking from the tissue via a channel formedby the needle. Such considerations are explored in more detail in U.S.Patent Application No. 2004/0191225—see also U.S. Pat. No.5,848,996—which are incorporated herein by reference in theirentireties. Nevertheless, the scope of the invention is not limited topencil point needles. Bevel-tipped and blunt-tipped needles can also beemployed according to various embodiments. In particular, the inventorshave conducted tests using prototypes with blunt-tipped needles, whichperformed satisfactorily.

FIG. 3 shows a solid-core delivery needle 130 having a plurality ofannular grooves 132. The needle 130 is a “passive delivery” device,meaning that a therapeutic agent is not delivered under pressure from areservoir but instead is carried into the tissue in the grooves 132.Other passive delivery-type needles include, for example, needles withmicro pits over their surfaces, needles coated with nanowire, andneedles made from porous materials. Such a needle is immersed in aliquid agent for sufficient time to charge, and is then inserted intothe target tissue. In the case of the needle 130 of FIG. 3, the needlecan be charged by being briefly dipped into the agent. A porous needlewill carry more of the agent, but may require more time to charge, andmay likewise need to be left in place in the tissue for a longer periodto deliver its charge.

Embodiments are primarily described herein as using active deliveryneedles, i.e., needles that actively force fluid into the surroundingtissue. However, passive delivery needles such as those described abovecan also be employed, according to the design parameters of a givenapplication.

FIGS. 4A and 4B show a portion of an inserter needle 140 and a portionof a delivery needle 120 similar to those described with reference toFIGS. 2A-2D. In FIG. 4A, the inserter needle 140 is positioned such thatthe tip-end 124 of the delivery needle 120 extends slightly beyond anend of the inserter needle 140. In this configuration, the needle 120and inserter needle 140 can be inserted through the skin of a subject,such as a patient or test model. The combination of the needle 120 andinserter needle 1406 are configured to have sufficient stiffness topenetrate the skin without bending, and the tapered point of the tip-end124 assists in the penetration. Additionally, the inserter needle 140covers the ports 122 of the needle 120 and prevents contamination of thecontents of the needle by non-target tissue, and vice-versa. When thetip-end 124 of the needle has penetrated to within a small distance ofthe target tissue, the inserter needle 140 is held in position whileinsertion of the needle 120 continues until it is correctly positionedin the target tissue. The insertion distance of the inserter needle 140can be selected such that the needle 120 is correctly positioned onceall of the ports 122 are clear of the inserter needle, as shown in FIG.2B. In this way, the needle 120 can be provided with maximum protectionand support, and the likelihood of contamination can be minimized.

According to an alternate embodiment, a stylette is positioned in theinserter needle to stiffen the needle and prevent collection of a tissueplug during insertion. Once the inserter needle 140 is positioned, thestylette is removed and the delivery needle 120 is inserted.

FIG. 5 is a diagrammatic view of a delivery assembly 150 according toanother embodiment. The delivery assembly 150 includes a needle array152, an inserter assembly 154, an actuator assembly 156, a driverassembly 158, a control assembly 160, and a frame 162. The frame 162provides a substantially rigid structure to which other elements of theassembly 150 are coupled.

The needle array 152 comprises a plurality of needle cylinders 166 and aneedle block 168. In the embodiment shown, the needle block 168 isintegral with the frame 162. Each of the plurality of needle cylinders166 is coupled, at a first end 170, in a respective needle aperture 174extending in the needle block 168, and comprises a lumen 176, having, inthe illustrated embodiment, a nominal diameter of 0.15 mm, extendingsubstantially the entire length of the needle cylinder 166. Each needlecylinder 166 includes a reservoir 178 in a region toward the first end170, a needle 120 in a region toward a second end 180, and a tip-end 124at the second end 180 of the needle cylinder 166. In the embodimentshown, the tip-end 124 is tapered to a point.

Each delivery needle 120 is defined by a plurality of ports 122distributed along its length. The length of each of the plurality ofneedle cylinders 166 and of the respective needles 120 varies accordingto the embodiment. In one embodiment, each needle cylinder 166 is longerthan 15 cm, while according to other embodiments the needle cylindersare each longer than 10 cm, between 5 cm and 10 cm, and as short as 2cm, respectively. Likewise, according to various embodiments, each ofthe plurality of delivery needles 120, defined by the portion of therespective needle cylinder 166 along which the ports 122 are spaced, islonger than 1 cm, longer than 2 cm, longer than 4 cm, and longer than 8cm.

The inserter assembly 154 comprises a plurality of inserter needles 140coupled to an inserter block 192 in respective inserter apertures 190extending therein in a configuration that corresponds to the arrangementof the needle cylinders 166 in the needle block 168, such that each ofthe plurality of needle cylinders 166 can be positioned within arespective one of the plurality of inserter needles 140 as shown in FIG.5. The inserter assembly 154 is axially slidable over the needlecylinders 166 between a first position, in which only the tip-ends 124of each of the needle cylinders 166 extend from respective ones of theplurality of inserter needles 140, to a second position, in which thesecond ends 180 of each of the needle cylinders 166 extends from therespective inserter needle 140 a distance sufficient to clear all of theports 122 of the respective delivery needle 120.

According to an embodiment, a spacer is provided, configured to bepositioned between the inserter block 192 and the needle block 168,sized such that when the inserter block and the needle block are bothengaged with the spacer, the inserter block is maintained in the firstposition. Removal of the spacer permits movement of the inserter block192 and the needle block 168 relative to each other, to permit placementof the inserter block into the second position, relative to the needleblock.

The actuator assembly 156 comprises a plurality of plungers 200 coupledat respective first ends 204 to a plunger block 206 in a configurationthat corresponds to the arrangement of the needle cylinders 166 and theinserter needles 140 such that a second end 208 of each of the pluralityof plungers 200 can be positioned within the reservoir 178 of arespective one of the plurality of the needle cylinders 166 as shown. AnO-ring 210 is provided at the second end 208 of each of the plurality ofplungers 200 to sealingly engage the wall of the respective lumen 176.The actuator assembly 156 also comprises an actuator 212 coupled to anactuator block 214, which in turn is rigidly coupled to the plungerblock 206. In the embodiment shown, the actuator 212 comprises amicrometer device 220 having a thimble 222, a barrel 224, and a spindle228 such as are well known in the art. The barrel 224 is rigidly coupledto the frame 162 while the spindle 228 is rotatably coupled to theactuator block 568 so as to control translational movement of theactuator block relative to the frame 162. The micrometer device 568 iscalibrated in 0.01 mm increments, with a spindle travel of 0.5 mm perrotation of the thimble 222 and a maximum stroke of 15 mm. Thus, eachcomplete rotation of the thimble moves each of the plurality of plungers0.5 mm within the lumen 178 of the respective needle cylinder 166 anddisplaces about 0.0001 cm³ of volume, or 0.1 nL per revolution. Thus,given a maximum stroke of 15 mm, the maximum dispensing capacity of eachof the plurality of needles 120 is about 3 nL.

The driver assembly 158 comprises a stepper motor 230 such as is wellknown in the art, and that includes a motor casing 232, a motor shaft234 coupled to a rotor of the motor 230, and other elements such as arewell known in the art. The motor casing 232 is rigidly coupled to theframe 162, and the motor shaft 234 is slidably coupled to the thimble222 of the micrometer device 568 while being rotationally lockedtherewith, such as via a spline coupling, for example. Accordingly,rotational force from the motor shaft 234 is transmitted to the thimble222, while axial movement of the thimble is not limited by the motorshaft. Such couplings are well known in the mechanical arts. The steppermotor 230 of the illustrated embodiment is configured to divide eachrotation into 125 steps. Thus, each incremental rotational step of themotor 230 rotates the thimble about 3°, displacing a volume of about 0.8pL per reservoir 178.

The controller assembly 160 includes a controller 240 and a controlcable 242 that extends from the controller to the stepper motor 230.Signals for controlling direction, speed, and degree of rotation of themotor shaft 234 are transmitted from the controller 240 to the steppermotor 230 via the control cable 242 in a manner that is well known inthe field to which such motors belong. According to an embodiment, thecontroller is programmable. A user can program the controller to controla speed of delivery of a fluid from the delivery needles 120 byselecting the speed of rotation, and a volume of fluid delivered byselecting the number of partial and complete rotations of the rotor.According to another embodiment, the controller is manually operated,such that a user controls a rate and direction of rotation of the motor230 in real time. According to a third embodiment, the driver andcontroller assemblies are omitted, and a user controls fluid delivery bymanually rotating the thimble 222 of the actuator assembly 212.

Charging the reservoirs 178 can be accomplished in a number of ways. Forexample, a charging vessel can be provided that includes a plurality ofcups or compartments in an arrangement that corresponds to thearrangement of the needle cylinders 166. The user first places aselected fluidic agent or combination of agents in each of the cups. Thedelivery assembly 150 of FIG. 5 is positioned with the needle cylinderspointing downward as shown in the drawing, and the spindle 228 of theactuator 212 fully extended. The frame 162 is lowered until the needles120 are fully immersed in the fluids in the respective cups. The motor230 is then controlled to rotate in the reverse direction, drawing thespindle 228 inward and pulling the plungers 200 upward. This in turncreates a negative pressure in the reservoirs 178 relative to ambient,drawing the fluids into the needle cylinders 166 via the needle ports122. When the reservoirs are sufficiently charged, rotation of the rotoris halted and the needle array 152 is withdrawn from the chargingvessel.

In order to deliver the charge, according to one embodiment, each of theneedle cylinders 166 of the needle array 152 is positioned in arespective one of the inserter needles 140 of the inserter assembly 154so that the tip-ends 178 of the needles 120 protrude from the inserterneedles 140, substantially as described with reference to FIG. 4A. Thedelivery assembly 150 is then positioned in axial alignment with atarget tissue region of a subject and translated axially so that thetip-ends of the needles 120 penetrate the subject's skin. Axialtranslation of the delivery assembly 150 continues until the tip-ends124 of the needle cylinders 166 have penetrated to within a selecteddistance of the target tissue region. The inserter assembly 154 is thenheld in position while the frame 162 and the elements coupled theretocontinue to move axially, such that the needles 120 extend into thetarget tissue region. When the needles 120 are correctly positioned,movement of the delivery assembly 150 is halted and the frame 162 isheld in position relative to the subject. The stepper motor 230 is thencontrolled to rotate the thimble 222 in the forward direction so as tocause the spindle 228 to extend, driving the plungers 200 into theneedle cylinders 166 and creating an overpressure in the respectivereservoirs 178, thereby forcing fluid from the reservoirs to the targettissue region via the ports 122 of the delivery needles 120.

Delivery can be performed in a few seconds, or it can be extended overminutes or hours under a relatively low overpressure to promote completeabsorption of the fluid into the surrounding tissue. According to theembodiment described with reference to FIG. 5, the stepper motor 230 canbe controlled to rotate the rotor fast enough to depress the plungers200 the full 15 mm in less than one second, or slow enough that a singlerotation can take many hours.

FIG. 6 shows a portion of a needle array 250 according to anotherembodiment. A portion of a needle cylinder 166 is shown, together with aportion of a needle block 252. The first end 170 of the needle cylinder166 is coupled to a first portion 254 of an aperture 256 extending inthe needle block 252. The aperture 256 includes the first portion 254,sized to receive the needle cylinder 166, and a second portion 258having an increased diameter. In the embodiment shown, the diameter ofthe second portion 258 has a diameter of 0.75 mm. The second portion 258defines a reservoir that is in fluid communication with the needlecylinder 166. The second end of a plunger 260 is positioned in thesecond portion 258, with an O-ring 262 sealingly engaged therein.

In the arrangement described with reference to the delivery assembly 150of FIG. 5, the second end of each of the plurality of plungers 200 ispositioned in a respective one of the needle cylinders 166, and thereservoirs 178 are comprised by the needle cylinders. Thus, axialmovement of one of the plurality of plungers 200 within the lumen 176 ofa respective needle cylinder 166 displaces a volume equal to atransverse cross-sectional area of the lumen, multiplied by the distanceof travel of the plunger. In contrast, because of the diameter of thesecond portion 258 of the aperture 256 of FIG. 6, relative to thediameter of the lumen 176, axial Movement of the plunger 260 displaces avolume that is greater than the volume displaced by a plunger 200 ofFIG. 5 by a factor of 25, for a given distance of travel. Conversely, todisplace an equal volume of the reservoir 178, a plunger 200 of FIG. 5must travel 25 times as far as the plunger 260 in the second portion 258of the aperture. Thus, given otherwise identical elements, the deliveryassembly 150 of FIG. 5 is capable of accurately metering delivery ofsmaller quantities of fluid than a similar assembly having reservoirsand plungers configured as described with reference to FIG. 6, while thelatter is capable of delivering larger quantities of fluid for a givenplunger stroke length; up to 75 nL over a stroke of 15 mm vs. 3 nL forthe embodiment of FIG. 5. Of course, the values given are exemplary; oneof ordinary skill will recognize that the needle sizes as well asreservoir sizes can be selected to accommodate particular requirements.

Turning now to FIG. 7, elements of a delivery assembly 270 are shownaccording to another embodiment. A needle block 272 includes a largeplurality of needle apertures 274 extending therethrough, arranged in aclosely spaced array. Needle cylinders 166 are provided separately, invarious assortments of lengths and numbers, sizes, and spacings ofports.

In use, a user selects a number of needles to be used for a particularprocedure, and selects the particular needle cylinders 166, placing eachin a respective one of the plurality of apertures 274 of the needleblock, in an arrangement that is selected for the particular procedure.The user may require only a small number of needles, such as one tofive, for example, or may require hundreds or thousands of needles.Furthermore, the needle cylinders 166 can be of varying lengths andconfigurations. The user selects the arrangement of the needle cylinders166 in the needle block 272, and their respective lengths andconfigurations, at least in part according to factors such as the size,shape, and position of a target tissue region in a subject's body, thedesired distribution density of fluid in the target tissue region, thepermeability of the target tissue, etc.

The needle cylinders 166 can be affixed in the apertures 274 by anyappropriate means, including, for example, soldering, brazing, and byadhesive. Alternatively, the needle cylinders 166 can be sized andconfigured to be fixed in place by an interference fit, such that, forexample, the first end of each needle cylinder has a fractionallyincreased outer diameter. The user drops each needle cylinder into arespective aperture, tip-end first, then pulls the needle cylinder intothe aperture from the other side of the needle block until the first endis firmly engaged in the aperture. A plunger block and an inserter blockare also provided, with respective pluralities of apertures in arrayscorresponding to the array of needle apertures of the needle block. Theuser loads plungers 200 and inserter needles 140 concurrently with theneedle cylinders 166 for operation in a delivery assembly similar tothat described with reference to FIG. 5. Provided the method used toattach the needle cylinders 166 in place can be reversed, the needleblock 272 can be reused repeatedly for different procedures.

The needle block 272 shown in FIG. 7 is about 5 cm in diameter, 1 cm inthickness, and has approximately 1,600 apertures, spaced about 1 mmapart. According to other embodiments, the needle block can be anyappropriate shape and size; from as small as 1 or 2 centimeters acrossto as large as ten or more centimeters across, and can have any numberof apertures, from ten or fewer to several thousand. According tovarious embodiments, the needle block is provided, for example, with200, 400, and 800 apertures. The large number of apertures providessignificant freedom to a user to control spacing between needles as wellas the particular pattern of the array. The needle block 272 is shownwith a hexagonal grid array of apertures. The apertures can also bearranged in other grid configurations, such as, for example, rectangularand quincunx. The needle cylinders shown are about 5 cm in length, butthis too is merely exemplary.

The delivery actuators of previous embodiments have been described asplungers. However, any suitable actuator can be used to control anamount of therapeutic agent delivered from the reservoirs into theneedle. For example, fluid pressure such as by compressed air orpressurized liquid can be used to control an amount of therapeutic agentdelivered to a region of biological tissue via the reservoirs andneedles.

Referring now to FIG. 8, a delivery assembly 300 is shown, according toanother embodiment. The delivery assembly 300 includes a plurality ofneedle cylinders 302 comprising respective reservoirs 178 and needles120. Fluid couplings 312 place the needle cylinders 302 in fluidcommunication with a manifold 304. A fluid pressure source 306 and afluid vacuum source 308 can each be placed in fluid communication withthe manifold 304 by operation of a valve 310.

According to the embodiment of FIG. 8, the needle cylinders 302 are notfixed with respect to each other, but can be individually emplaced, in atarget tissue region, for example. The reservoirs are first charged, byplacing the delivery needles 120 in a selected fluid, e.g., atherapeutic agent or respective therapeutic agent, and the fluid vacuumsource is placed in fluid communication with the manifold, drawing anegative pressure into the reservoirs and drawing the agent into theneedles. The user then positions the needles 120 in the target tissueregion. When they are all in place, the manifold 304 is pressurized,forcing fluid from the reservoirs of each of the needle cylinders 166via the ports 122 of the respective delivery needles. While FIG. 8 showsa simple fluid circuit, it will be understood that in practice such acircuit could include any of valves, pressure regulator, peristalticpump, microfluidic pump, vacuum accumulator, compressor, controller,etc., all of which are well known in the art, and within the abilitiesof one of ordinary skill to select and configure for a givenapplication.

According to an embodiment, solid tissue into which a plurality oftherapeutic agents have been delivered is subsequently resected from thesubject and evaluated. For example, in a case where the target tissue isa cancerous tumor, the plurality of agents injected therein can includesome agents whose efficacy or effect on such tumors is underinvestigation. By injecting the various agents in vivo then waiting aselected period before removing the tumor, the effect of the agents onthe tumor in situ can be investigated. This preserves the tumormicroenvironment and distinguishes this method from current ex vivo orin vitro therapeutics evaluation methods. Assuming that the needles usedare configured to deliver a substantially equal amount of fluid at anygiven location along their length, as described above with reference toFIGS. 2B-2D, the agent delivered by each of the needles is evenlydistributed to the surrounding tissue along the delivery axis on whichthe respective needle 120 was positioned during the delivery of theagent to the tumor 320. Over time, each agent permeates outward from itsdelivery axis to a greater or lesser degree, depending on factors suchas, for example, the density of the surrounding tissue, the viscosityand composition of the agent, the wettability of the tissue by therespective agent, etc. Typically, the portions of the tissue into whichthe agents spread are approximately column-shaped regions coaxial withthe respective delivery axes.

According to various embodiments, a region of tissue is left in placefor some period of time before being resected. For example, 48-72 hoursfollowing delivery is thought to be generally sufficient for a tumor toexhibit a detectable response. In other cases, the wait period may behours, days, or weeks. According to some embodiments, the tissue regionis imaged using known methods to precisely locate the target region oftissue prior to insertion of the needles. The region may be imagedrepeatedly before and after delivery of the plurality of agents to theregion of tissue.

According to other embodiments, a plurality of agents are delivered to aportion of tissue via respective ones of a plurality of needles of aneedle array after the portion of tissue is resected.

Referring now to FIG. 9, a portion of a tumor 320 is shown, following aninjection procedure and subsequent resection. The tumor 320 has beensectioned into a plurality of slices 322 along planes that liesubstantially normal to the delivery axes. Column-shaped deliveryregions 324 define the regions of permeation of the respective agents,and extend perpendicular to the planes of the sections 322.

Many of the regions 324 may not be easily detectable to a user, sogenerally at least two readily detectable position markers 324 a, 324 bare among the agents injected, at widely separated locations. The usercan then overlay a template on which the locations of each of thedelivery axes is marked, aligning the indicated marker positions of thetemplate with the detectable position markers 324 a, 324 b of a givensection 322, thereby locating the remaining delivery regions 324. Theposition markers 324 a, 324 b can be any composition that is detectableby a user. Various exemplary position markers are described in detailelsewhere in this disclosure. According to an embodiment, the positionmarkers are selected to resist permeation and diffusion into thesurrounding tissue and to remain concentrated in a narrow column, asshown for example at 324 a, so as to be detectable for an extendedperiod after the injection procedure, and to provide an accurate guidefor positioning the template.

In addition to position markers, control agents may also be among theagents injected. For example, a negative control can comprise asubstance used as a vehicle in others of the agents, and a positivecontrol can comprise a compound of most or all of the agents deliveredindividually at other delivery axes.

Following sectioning of the tumor 320, a user conducts selected assayson delivery regions 324 of various sections 322 of the tumor 320, asdescribed in more detail later. One benefit of the devices and methodsdisclosed herein is that, in addition to evaluating the efficacy of agiven agent on the tumor, the efficacy of agents at various deliveryregions 324 can be evaluated and compared. Additionally, the effect of agiven agent on various parts of the tumor can be evaluated, bothvertically and horizontally. By comparing the effect of an agent in adelivery region 324 c at section 322 a, for example, with its effect inthe same region 324 c at sections 322 b and 322 c, the effect of thatagent on different tissue compositions that may occur vertically can bedifferentiated. Similarly, the same agent can be delivered at severaldelivery axes in the array, e.g., 324 c and 324 d, and the relativeeffects at those locations in a given section 322 can then be compared,providing horizontal differentiation. As is well known in the art,biological tissue is rarely homogeneous over even relatively smalldistances. A given agent might have substantially no effect on sometissue structures of a tumor, but might, on the other hand, be extremelyeffective on others. Such differential effects can be detected andevaluated as described above.

Another valuable aspect that can be evaluated is the effect of multipleagents in regions where they interact within the tissue. Deliveryregions 324 e and 324 f are spaced more closely together than theothers, resulting in the respective agents interacting in a region 324ef where the respective delivery regions overlap.

As discussed in the background section of this disclosure, clinicaltrials for cancer related therapeutics are incredibly expensive and timeconsuming. It is therefore very important to effectively screen foragents that have relatively greater potential as early in the process aspossible. Agents subjected to such screening are sometimes referred toas candidate effective agents. One screening method involves placingeach candidate agent in a respective Petri dish with a growth medium. Acancerous tumor is reduced to a homogeneous slurry and is distributedamong the Petri dishes and incubated. The dishes are later evaluated forindications of cell growth. Agents that appear to have impeded growth ofcancer cells may then be advanced for further study.

However, this method is only marginally effective, for several reasons.First, many cancers are known to be nonviable outside a live subject,for reasons such as lack of a blood supply, etc., and fail to grow invitro under any circumstances. Screening tests like that described aretherefore ineffective with these. In some cases it is not known that aparticular strain falls in this category prior to conducting the test.The result is that an expensive and time consuming test is inconclusive,and the tumor cannot be salvaged for an alternative test. If the tumoris of a rare strain, it may be sometime before another is available foralternative testing.

Second, the reduction process can alter the response characteristics ofa tumor. The process involves essentially pureeing the tumor, whichcompletely destroys any structural differentiation, and may render thecancer susceptible to some agents that would have no effect on the samestrain in vivo, resulting in a false positive, even though such agentsmight be useless for treating the cancer in patients. The result is thatmany such agents are not eliminated until later phases of study, aftermuch more money and effort have been expended.

Third, the same reduction process can also produce false negatives, inwhich some agents may fail to inhibit cell growth in vitro, but would beeffective in treating the same cancer in vivo. This results in thepremature elimination of some agents that might otherwise have becomeeffective therapeutic options.

Many false positives or false negatives are generated in the current exvivo or in vitro art because tumor cells are separated from theirmicroenvironment, e.g., surrounding non-cancerous cells, blood,hormones, paracrine factors, oxygen tension, cell-cell communications,and host immune functions, all of which may influence whether certaintherapeutic agents have or do not have activity in cancer cells.

Fourth, even where accurate, only the most general information can begleaned from such studies because the test conditions do not remotelyresemble the conditions in which the cancer normally lives and grows,and in which it is treated therapeutically.

It is also known to inject a test agent into a tumor prior to resectionfrom a subject, for subsequent examination. However, in such tests asingle agent is typically injected, so they are not feasible for earlyscreening, but are usually reserved for agents that have alreadydemonstrated significant potential. Even in animal models, it isexpensive and time consuming to induce a tumor and allow it to grow to apractical size, which makes extensive early screening by this methodimpractical.

Finally, even where general efficacy of an agent in treating aparticular cancer type, subtype, variant, strain or the like has beendemonstrated, it is not uncommon for the cancer of a particular patientto be wholly unresponsive to the agent. The patient is thus exposed tothe often extreme discomfort and toxicity of the treatment—not tomention the cost—without significant benefit. Worse, because the agent'sineffectiveness may not be known for a long period while the treatmentis ongoing, the opportunity to shift to a different treatment that mighthave been completely successful may be lost.

Where a similar idiosyncratic response—or lack thereof—occurs in asubject of a drug study, the results of the study can be skewed. Toavoid this, it is typically necessary to resort to larger test groups tominimize the statistical impact of nonresponding study subjects, whichfurther increases the cost of such studies.

The inventors have recognized that the inability in the known art toaccurately position an agent in tissue in vivo, especially with respectto other agents, and the inability to later identify the locations ofagents in tissue, prevent more extensive and beneficial use of in vivoinjection, and likewise, that if such accuracy could be achieved,significant benefits in research and therapy could be realized.

It has been noted above that the volume of fluid that is delivered byeach delivery needle can be vanishingly small, much less than would be aminimal dose required to produce a detectable effect in an adult.Depending on the agent, the effect may nevertheless be detected on thevery small region immediately surrounding the delivery site.Accordingly, candidate effective agents can be injected into a tumor,for example, in situ, without danger of harming the subject.Additionally, a significant number of different agents can besimultaneously delivered to respective delivery axes within the tumor

The procedures described above can be employed to resolve a number ofthe problems and difficulties that contribute to the cost and delay ofdeveloping effective cancer therapies. For example, because thecandidate agents are delivered in vivo, the tumor is not otherwisedisturbed, and so its reaction to each agent will tend to be indicativeof its reaction if exposed to that agent in therapeutically effectivequantities. The incidence of false positives and false negatives issignificantly reduced.

Second, because relatively large numbers of agents can be delivered to atumor without significant danger to the subject, it is practical to usethe procedure to screen large numbers of candidate agents early in thetesting process, perhaps eliminating those that show the least promise,flagging the most promising agents for additional study, or prioritizingcandidates for further study.

Third, again because of the large number of agents that can be deliveredto a tumor, potential study subjects can be screened for response toparticular agents, reducing or eliminating the number of subjects withidiosyncratic responses.

Fourth, when employed in a therapeutic setting, a patient can be testedfor response to a large number of treatments and the most promising canbe identified early in the process, thereby reducing the number ofpatients who undergo ineffective treatments and improving the likelihoodthat a patient will receive the most effective available treatment.

According to an embodiment, a plurality of candidate effective agentsare delivered, in vivo, along mutually parallel axes to a region ofsolid tissue, substantially as described above. The region of solidtissue is subsequently resected from a subject and an effect of each ofthe plurality of agents on the solid tissue is evaluated. Based on theevaluation, one or more of the plurality of agents are prioritized forfurther investigation.

According to alternate embodiments, one or more of the plurality ofagents are selected or deselected for further investigation, based onthe evaluation.

According to another embodiment a plurality of agents that have beenshown to have therapeutic benefits are delivered, in vivo, alongmutually parallel axes to a region of solid tissue. The region of solidtissue is subsequently resected from a subject and an effect of each ofthe plurality of agents on the solid tissue is evaluated. Based on theevaluation, one or more of the plurality of agents are prioritized fortherapeutic treatment of the subject. According to alternateembodiments, one or more of the plurality of agents are selected ordeselected for therapeutic treatment of the subject, based on theevaluation.

According to another embodiment a plurality of agents that are currentlyunder investigation for therapeutic efficacy are delivered, in vivo,along mutually parallel axes to a region of solid tissue in each of aplurality of subjects. The respective regions of solid tissue aresubsequently resected from each of the subjects and an effect of each ofthe plurality of agents on the respective regions of solid tissue isevaluated. Based on the evaluation, one or more of the plurality ofsubjects are selected as candidates for further study of the therapeuticefficacy of one or more of the agents. According to alternateembodiments, one or more of the plurality of subjects are deselected ascandidates for further study of the therapeutic efficacy of one or moreof the agents.

According to some embodiments, needle arrays are employed to effectivelyand broadly deliver therapeutic agents to live and viable tissue (e.g.,solid tissue) in a subject, wherein imaging is not required and thetissue is not subsequently resected. The subject may be monitoredaccording to appropriate clinical criteria for assessing clinicalimprovement. In these and related embodiments, it will be appreciatedthat significantly higher levels of the therapeutic agent will beachieved within the solid tissue than would be the case if the agentwere delivered systemically, although detectable amounts of thetherapeutic agent may be subsequently identified outside the solidtissue (e.g., in the circulation). As a non-limiting example, one suchembodiment contemplates direct intramuscular introduction of a genetherapy agent for treating muscular dystrophy (e.g., an engineeredtherapeutic virus, a therapeutic agent-carrying nanoparticle, etc.) toone or more skeletal muscle injection sites in a subject, without theneed for imaging, surgery, or histology on biopsy specimens. Of course,periodic monitoring of the circulation for leaked therapeutic agentand/or subsequent analysis of a biopsy specimen, e.g., to assess theeffects of the agent on the target tissue, may also be considered.

In other embodiments it is contemplated that the target region in asolid tissue may be imaged using known techniques to evaluate theeffects of the agents. The imaging can be by any suitable process ormethod, including, for example, radiographic imaging, magnetic resonanceimaging, positron emission tomogoraphy, biophotonic imaging, etc. Insome embodiments, the target region may be imaged repeatedly before,during, and after the delivery process.

According to the embodiment of FIG. 10, a data processing system 350 isused to carry out or direct operations, and includes a processor 354 anda memory 356. The processor 354 communicates with the memory 356 via anaddress/data bus 360 and also communicates with a needle array assembly362 and a patient scanning device 364. The patient scanning device 364is used, according to an embodiment, to assist in placing the needles ofthe needle array assembly 362 in a patient in vivo and for non-invasiveanalysis of target tissue regions using imaging techniques, such asradiographic imaging or nuclear medical assays. The processor 354 can bea commercially available or custom microprocessor, microcontroller,signal processor or the like. The memory 356 can include any memorydevices and/or storage media containing the software and data used toimplement the functionality circuits and modules.

The memory 356 can any of include several categories of software anddata used in the data processing system, such as, for example, anoperating system 366, application programs 368; input/output devicedrivers 370; and data 372. The application programs 368 are illustrativeof the programs that implement the various features of the circuits andmodules according to some embodiments, and the data 372 represents thestatic and dynamic data used by the application programs 368, theoperating system 366, the input/output device drivers 370 and othersoftware programs that may reside in the memory 356.

According to various embodiments, the data processing system 350 mayinclude several modules, including an array controller 376, a scannercontroller 378 and the like. The modules may be configured as a singlemodule or additional modules otherwise configured to implement theoperations described herein. For example, the array controller 376 canbe configured to control the needle array assembly 100 of FIG. 1, bycontrolling the actuators 116, and consequently, the release oftherapeutic agents from the reservoirs 114 via the needles 112. Thescanner controller 378 can be configured to control the patient scanningdevice 364.

Certain embodiments described herein relate to introducing an agent intoa solid tissue in a subject, and/or excising all or a portion of a solidtissue from a subject, and/or obtaining one or more biological samplesfrom a solid tissue that may be in a subject, and/or screening one ormore subjects for clinical trial eligibility, and/or any number of othermethods that may involve a subject, which includes a subject orbiological source.

The subject or biological source may be a human or non-human animal, atransgenic or cloned or tissue-engineered (including through the use ofstem cells) organism, a primary cell culture or culture adapted cellline including but not limited to genetically engineered cell lines thatmay contain chromosomally integrated or episomal recombinant nucleicacid sequences, immortalized or immortalizable cell lines, somatic cellhybrid cell lines, differentiated or differentiatable cell lines,transformed cell lines and the like. In certain preferred embodiments ofthe invention, the subject or biological source may be suspected ofhaving or being at risk for having a malignant condition, and in certainpreferred embodiments of the invention the subject or biological sourcemay be known to be free of a risk or presence of such disease.

Certain preferred embodiments contemplate a subject or biological sourcethat is a human subject such as a patient that has been diagnosed ashaving or being at risk for developing or acquiring cancer according toart-accepted clinical diagnostic criteria, such as those of the U.S.National Cancer Institute (Bethesda, Md., USA) or as described inDeVita, Hellman, and Rosenberg's Cancer: Principles and Practice ofOncology (2008, Lippincott, Williams and Wilkins, Philadelphia/Ovid, NewYork); Pizzo and Poplack, Principles and Practice of Pediatric Oncology(Fourth edition, 2001, Lippincott, Williams and Wilkins,Philadelphia/Ovid, New York); and Vogelstein and Kinzler, The GeneticBasis of Human Cancer (Second edition, 2002, McGraw Hill Professional,New York); certain embodiments contemplate a human subject that is knownto be free of a risk for having, developing or acquiring cancer by suchcriteria.

Certain other embodiments contemplate a non-human subject or biologicalsource, for example a non-human primate such as a macaque, chimpanzee,gorilla, vervet, orangutan, baboon or other non-human primate, includingsuch non-human subjects that may be known to the art as preclinicalmodels, including preclinical models for solid tumors and/or othercancers. Certain other embodiments contemplate a non-human subject thatis a mammal, for example, a mouse, rat, rabbit, pig, sheep, horse,bovine, goat, gerbil, hamster, guinea pig or other mammal; many suchmammals may be subjects that are known to the art as preclinical modelsfor certain diseases or disorders, including solid tumors and/or othercancers (e.g., Talmadge et al., 2007 Am. J. Pathol. 170:793; Kerbel,2003 Canc. Biol. Therap. 2(4 Suppl 1):S134; Man et al., 2007 Canc. Met.Rev. 26:737; Cespedes et al., 2006 Clin. Transl. Oncol. 8:318 ). Therange of embodiments is not intended to be so limited, however, suchthat there are also contemplated other embodiments in which the subjector biological source may be a non-mammalian vertebrate, for example,another higher vertebrate, or an avian, amphibian or reptilian species,or another subject or biological source.

Biological samples may be provided by obtaining a blood sample, biopsyspecimen, tissue explant, organ culture, biological fluid or any othertissue or cell preparation from a subject or a biological source. Incertain preferred embodiments the biological sample may be obtained froma solid tissue (e.g., a solid tumor) using the herein described device,for example, by introducing a multiple needle device into a solidtissue, thereby placing a plurality of needles at a plurality ofpositions in the tissue, and generating negative pressure at one or aplurality of ports of each needle of the multiple needle device underconditions and for a time sufficient to draw into the needles aplurality of biological samples from the plurality of positions in thetissue.

Devices and methods disclosed here may find uses according to certainpreferred embodiments for the introduction of agents to, and/or thewithdrawal of biological samples from, a solid tissue, which may bepresent in a subject in vivo including a solid tissue that may beaccessed further to a surgical procedure, or that may be excised, forinstance incident to a surgical procedure according to standard medicalpractices.

Solid tissues are well known to the medical arts and may include anycohesive, spatially discrete non-fluid defined anatomic compartment thatis substantially the product of multicellular, intercellular, tissueand/or organ architecture, such as a three-dimensionally definedcompartment that may comprise or derive its structural integrity fromassociated connective tissue and may be separated from other body areasby a thin membrane (e.g., meningeal membrane, pericardial membrane,pleural membrane, mucosal membrane, basement membrane, omentum,organ-encapsulating membrane, or the like). Non-limiting exemplary solidtissues may include brain, liver, lung, kidney, prostate, ovary, spleen,lymph node (including tonsil), thyroid, pancreas, heart, skeletalmuscle, intestine, larynx, esophagus and stomach. Anatomical locations,morphological properties, histological characterization, and invasiveand/or non-invasive access to these and other solid tissues are all wellknown to those familiar with the relevant arts.

Certain particularly preferred embodiments as disclosed herein relate toa method for selective delivery of a fluid-phase agent to a solidtissue. As also noted above, such selective delivery obviates the needfor excessive systemic concentrations of therapeutic or candidate agentsin order to achieve therapeutically effective concentrations in thedesired solid tissue, thereby avoiding clinically detrimental toxicitiesto uninvolved tissues and also avoiding undesirable side-effects.Related embodiments contemplate the testing of currently non-approvedcandidate agents through such selective delivery to a solid tissue.Without wishing to be bound by theory, according to these embodiments,direct effects of the candidate agent on the solid tissue (e.g., solidtumor) can be evaluated by in vivo administration followed by ex vivoanalysis of excised tissue, without threatening the health of thesubject, because the dose used for direct administration into the solidtissue is far lower than the minimal dose that would otherwise beadministered systemically. (The minimal dose is the smallest amount ofthe agent that will produce a desired physiologic effect in thesubject.) Given the minute volumes and low pressures of the presentmodes of fluid administration, and full or partial patency of the solidtissue as a physical property that promotes retention of theadministered fluid (also determinable by existing methodologies, e.g.,by imaging and/or by use of a detectable label as a tracer), the agentthat is selectively administered to the solid tissue according to thepresent disclosure is either undetectable outside the solid tissue, orif detectable outside the solid tissue, the agent is present at less (ina statistically significant manner) than the minimal dose.

Such considerations pertain in related embodiments, wherein detection ina solid tissue of an altered physiologic state subsequent to introducingan agent or a plurality of agents includes detecting a degree ofpermeation of the agent(s) through the solid tissue, detecting a degreeof absorption of the agent(s) in the tissue, detecting a physicochemicaleffect of the agent(s) on the tissue, and/or detecting a pharmacologicaleffect of the agent(s) on the tissue. Assays, including fluorescenceassays, of drug permeation or penetration in solid tissues are known inthe art and have been described (e.g., Kerr et al., 1987 Canc.Chemother. Pharmacol. 19:1 and references cited therein; Nederman etal., 1981 In Vitro 17:290; Durand, 1981 Canc. Res. 41:3495; Durand, 1989JNCI 81:146; Tunggal et al., 1999 Clin. Canc. Res. 5:1583) and may beconfigured further according to the present disclosure, for instance,through the detection in histological serial sections of a detectablelabel that has been co-administered to the solid tissue, prior toexcision and sectioning, with an agent of interest.

In such embodiments, permeation or penetration refers to the area ofretention of an agent in the solid tissue in the immediate vicinity ofthe needle from which the agent was introduced exclusive of perfusion(entry into and dispersion via any blood vessel), and may includeretention of the agent in extracellular space or extracellular matrix orin association with a cell membrane or intracellularly. Permeation maybe distinct from a physicochemical effect, which refers tomicroscopically detectable mechanical disruption of tissue that resultsfrom the needle insertion or fluid injection itself, or fromnon-biological mechanical or chemical tissue disruption caused by theagent (e.g., damage to cell membranes or disintegration of cell-celljunctions). Pharmacological effects include statistically significantalterations of a cell or tissue physiological state that are detectableas consequences of the molecular mechanism of action of the agent, forexample, cytoskeletal reorganization, extension or withdrawal ofcellular processes, or evidence of biological signal transduction as maybe detected using any of a number of known cytological, biochemical,molecular biological or other read-outs. Comparison of serial sectionsmay permit distinguishing the nature of the effect that is detectedhistologically.

Particularly preferred embodiments include those in which the solidtissue comprises a tumor, wherein agent delivery may be made to, and/orsample retrieval may be made from, the solid tumor. It will beappreciated by persons familiar with the art from the disclosure hereinthat in the course of practicing certain embodiments described herein, aselected region of a tumor may comprise the site into which the needlesof the presently described devices are inserted, introduced or otherwisecontacted with the tumor. The region may be selected on any number ofbases, including based on imaging that may be conducted before, duringor after a step of needle insertion, introduction or contacting, orbased on imaging conducted before, during or after excising the solidtissue from a subject, or based on other criteria including but notlimited to anatomic location, accessibility in the course of a surgicalprocedure, degree of vascularization or other criteria.

Solid tumors of any type are contemplated as being suitable forintervention using the devices described herein. In certain preferredembodiments, the solid tumor may be a benign tumor or a malignant tumor,which may further be a primary tumor, an invasive tumor or a metastatictumor. Certain embodiments contemplate a solid tumor that comprise oneof a prostate cancer cell, a breast cancer cell, a colon cancer cell, alung cancer cell, a brain cancer cell and an ovarian cancer cell, butthe invention is not intended to be so limited and other solid tumortypes and cancer cell types may be used. For example, the tumor maycomprise a cancer selected from adenoma, adenocarcinoma, squamous cellcarcinoma, basal cell carcinoma, small cell carcinoma, large cellundifferentiated carcinoma, chondrosarcoma and fibrosarcoma, or thelike. As also noted elsewhere herein, art-accepted clinical diagnosticcriteria have been established for these and other cancer types, such asthose promulgated by the U.S. National Cancer Institute (Bethesda, Md.,USA) or as described in DeVita, Hellman, and Rosenberg's Cancer:Principles and Practice of Oncology (2008, Lippincott, Williams andWilkins, Philadelphia/Ovid, New York); Pizzo and Poplack, Principles andPractice of Pediatric Oncology (Fourth edition, 2001, Lippincott,Williams and Wilkins, Philadelphia/Ovid, New York); and Vogelstein andKinzler, The Genetic Basis of Human Cancer (Second edition, 2002, McGrawHill Professional, New York). Other non-limiting examples of typing andcharacterization of particular cancers are described, e.g., inIgnatiadis et al, (2008 Pathobiol. 75:104); Kunz (2008 Curr. DrugDiscov. Technol. 5:9); and Auman et al. (2008 Drug Metab. Rev. 40:303).

An “altered physiologic state” may be any detectable parameter thatdirectly relates to a condition, process, pathway, dynamic structure,state or other activity in a solid tissue (and in preferred embodimentsin a solid tumor) including in a region thereof or a portion therefrom,further including a biological sample obtained therefrom, and thatpermits detection of an altered (e.g., measurably changed in astatistically significant manner relative to an appropriate control)structure or function in a biological sample from a subject orbiological source. The methods of the present invention thus pertain inpart to such correlation where an indicator of altered physiologic statemay be, for example, a cellular or biochemical activity, including asfurther non-limiting examples, cell viability, cell proliferation,apoptosis, cellular resistance to anti-growth signals, cell motility,cellular expression or elaboration of connective tissue-degradingenzymes, cellular recruitment of angiogenesis, or other criteria asprovided herein.

“Altered physiologic state” may refer to any condition or function whereany structure or activity that is directly or indirectly related to asolid tissue function has been changed in a statistically significantmanner relative to a control or standard, and may have its origin indirect or indirect interactions between a solid tissue constituent andan introduced agent, or in structural or functional changes that occuras the result of interactions between intermediates that may be formedas the result of such interactions, including metabolites, catabolites,substrates, precursors, cofactors and the like.

Additionally, altered physiologic state may include altered signaltransduction, respiratory, metabolic, genetic, biosynthetic or otherbiochemical or biophysical activity in some or all cells or tissues of asubject or biological source, in preferred embodiments in some or allcells of a solid tissue, and in more preferred embodiments in some orall cells of a tumor such as a solid tumor in a solid tissue. Asnon-limiting examples, altered biological signal transduction, cellviability, cell proliferation, apoptosis, cellular resistance toanti-growth signals, cell motility, cellular expression or elaborationof connective tissue-degrading enzymes, cellular recruitment ofangiogenesis, or other criteria including induction of apoptoticpathways and formation of atypical chemical and biochemical crosslinkedspecies within a cell, whether by enzymatic or non-enzymatic mechanisms,may all be regarded as indicative of altered physiologic state. Certainof these and other non-limiting examples are described in greater detailherein.

According to certain presently contemplated embodiments, the efficacy ofa candidate agent may be identified by detecting an altered physiologicstate as provided herein, including by assessing any of a number ofbiological parameters characteristic of a cancer cell such as thosereviewed by Hanahan and Weinberg (2000 Cell 100:57) and in thereferences cited therein. Therein are disclosed methodologies fordetermining the effect of a candidate agent on one or more traitsexhibited by cancer cells, and detectable by any of a variety oftechniques known to the art for determining one or more of (i) anability to evade apoptosis, (ii) acquisition of self-sufficiency ingrowth signals, (iii) insensitivity to growth-inhibitory signals, (iv)acquisition of tissue invasive and metastatic phenotype, (v) unlimitedreplicative potential, and (vi) sustained angiogenesis. Persons skilledin the art are familiar with multiple approaches for detecting thepresence of these alterations of physiologic state, which can be adaptedto a particular excised tumor system. See, e.g., Bonificano et al.(Eds.) Current Protocols in Cell Biology, 2007 John Wiley & Sons, NY;Ausubel et al. (Eds.) Current Protocols in Molecular Biology, 2007 JohnWiley & Sons, NY; Coligan et al. (Eds.), Current Protocols inImmunology, 2007 John Wiley & Sons, NY; Robinson et al. (Eds), CurrentProtocols in Cytometry, 2007 John Wiley & Sons, NY. Non-limitingexamples of parameters that may be assayed to identify an alteredphysiologic state include assays of cell viability, cell division,apoptosis, necrosis, cell surface marker expression, cellular activationstate, cellular elaboration of extracellular matrix (ECM) components orof ECM-degrading enzymes, morphometric analysis, extension or retractionof cellular processes, cytoskeletal reorganization, altered geneexpression, e.g., by in situ hybridization of immunohistochemistry(e.g., Shibata et al., 2002 J. Anat. 200:309) intracellularphosphoprotein localization (e.g., Gavet et al., 1998 J Cell Sci111:3333), and the like.

Selection of agents that are known or candidate oncology agents isunderstood and determinable by one skilled in the relevant arts (see,e.g., Berkowet al., eds., The Merck Manual, 16^(th) edition, Merck andCo., Rahway, N.J., 1992; Goodman et al., eds., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10^(th) edition, Pergamon Press,Inc., Elmsford, N.Y., (2001); De Vita, Hellman, and Rosenberg's Cancer:Principles and Practice of Oncology (2008, Lippincott, Williams andWilkins, Philadelphia/Ovid, New York); Pizzo and Poplack, Principles andPractice of Pediatric Oncology (Fourth edition, 2001, Lippincott,Williams and Wilkins, Philadelphia/Ovid, New York); Avery's DrugTreatment: Principles and Practice of Clinical Pharmacology andTherapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins,Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co.,Boston, (1985); Osolci al., eds., Remington's Pharmaceutical Sciences,18^(th) edition, Mack Publishing Co., Easton, Pa. (1990); Katzung, Basicand Clinical Pharmacology, Appleton and Lange, Norwalk, Conn. (1992)).Candidate agents may be selected from resources that disclose listingsof investigational therapeutics, for instance, the National Institutesof Health (Bethesda, Md.) which maintains a database of ongoing andplanned clinical trials at its “ClinicalTrials.gov” website.

Candidate agents for use in screening methods and in methods of ratingcandidate agents for development into therapeutic agents such as atherapeutic agent for treating a solid tumor may be provided as“libraries” or collections of compounds, compositions or molecules. Suchmolecules typically include compounds known in the art as “smallmolecules” and having molecular weights less than 10⁵ daltons,preferably less than 10⁴ daltons and still more preferably less than 10³daltons.

For example, a plurality of members of a library of test compounds canbe introduced as candidate agents to a region of a solid tumor of knowntumor type in each one or a plurality of subjects having a tumor of theknown tumor type, by distributing each of the candidate agents to aplurality of positions along an axis within the region in each subject,and after a selected period of time (e.g., a range of time, a minimumtime period or a specific time period) the region of solid tumor inwhich the candidate agents have been introduced can be imaged or removedfrom each subject, and each region compared by detecting an effect (ifany) of each agent on the respective position within the region, forinstance, by determining whether an altered physiologic state is presentas provided herein, relative to positions in the region that are treatedwith control agents as provided herein, which would either produce noeffect (negative control) or a readily detectable effect (positivecontrol).

Candidate agents further may be provided as members of a combinatoriallibrary, which preferably includes synthetic agents prepared accordingto a plurality of predetermined chemical reactions performed in aplurality of reaction vessels. For example, various starting compoundsmay be prepared employing one or more of solid-phase synthesis, recordedrandom mix methodologies and recorded reaction split techniques thatpermit a given constituent to traceably undergo a plurality ofpermutations and/or combinations of reaction conditions. The resultingproducts comprise a library that can be screened followed by iterativeselection and synthesis procedures, such as a synthetic combinatoriallibrary of peptides (see e.g., PCT/US91/08694, PCT/US91/04666, which arehereby incorporated by reference in their entireties) or othercompositions that may include small molecules as provided herein (seee.g., PCT/US94/08542, EP 0774464, U.S. Pat. No. 5,798,035, U.S. Pat. No.5,789,172, U.S. Pat. No. 5,751,629, which are hereby incorporated byreference in their entireties). Those having ordinary skill in the artwill appreciate that a diverse assortment of such libraries may beprepared according to established procedures, and tested for theirinfluence on an indicator of altered mitochondrial function, accordingto the present disclosure.

Other candidate agents may be proteins (including therapeutic proteins),peptides, peptidomimetics, polypeptides, and gene therapy agents (e.g.,plasmids, viral vectors, artificial chromosomes and the like containingtherapeutic genes or polynucleotides encoding therapeutic products,including coding sequences for small interfering RNA (siRNA), ribozymesand antisense RNA) which in certain further embodiments may comprise anoperably linked promoter such as a constitutive promoter or aregulatable promoter, such as an inducible promoter (e.g.,IPTG-inducible), a tightly regulated promoter (e.g., a promoter thatpermits little or no detectable transcription in the absence of itscognate inducer or derepressor) or a tissue-specific promoter.Methodologies for preparing, testing and using these and related agentsare known in the art. See, e.g., Ausubel (Ed.), Current Protocols inMolecular Biology (2007 John Wiley & Sons, NY); Rosenzweig and Nabel(Eds), Current Protocols in Human Genetics (esp. Ch. 13 therein,“Delivery Systems for Gene Therapy”, 2008 John Wiley & Sons, NY); Abell,Advances in Amino Acid Mimetics and Peptidomimetics, 1997 Elsevier, NY.

Other candidate agents may be antibodies, including naturally occurring,immunologically elicited, chimeric, humanized, recombinant, and otherengineered antigen-specific immunoglobulins and artificially generatedantigen-binding fragments and derivatives thereof, such as single-chainantibodies, minibodies, Fab fragments, bi-specific antibodies and thelike. See, e.g., Coligan et al. (Eds.), Current Protocols in Immunology(2007 John Wiley & Sons, NY); Harlow and Lane, Antibodies: A LaboratoryManual (1988 Cold Spring Harbor Press, Cold Spring Harbor, N.Y.); Harlowand Lane, Using Antibodies (1999 Cold Spring Harbor Press, Cold SpringHarbor, N.Y.).

Pharmaceutically acceptable carriers for therapeutic use are well knownin the pharmaceutical art, and are described, for example, in RemingtonsPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).For example, sterile saline and phosphate-buffered saline atphysiological pH may be used. Preservatives, stabilizers, dyes and otherancillary agents may be provided in the pharmaceutical composition. Forexample, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id. “Pharmaceuticallyacceptable salt” refers to salts of drug compounds derived from thecombination of such compounds and an organic or-inorganic acid (acidaddition salts) or an organic or inorganic base (base addition salts).The agents, including drugs, contemplated for use herein may be used ineither the free base or salt forms, with both forms being considered asbeing within the scope of the certain present invention embodiments.

The pharmaceutical compositions that contain one or more agents may bein any form which allows for the composition to be administered to apatient. According to certain preferred embodiments the composition willbe in liquid form and the route of administration will compriseadministration to a solid tissue as described herein. The termparenteral as used herein includes transcutaneous or subcutaneousinjections, and intramuscular, intramedullar and intrasternaltechniques.

The pharmaceutical composition is formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a subject such as a human patient. Compositions thatwill be administered to a patient may take the form of one or more dosesor dosage units, where for example, a pre-measured fluid volume maycomprise a single dosage unit, and a container of one or morecompositions (e.g., drugs) in liquid form may hold a plurality of dosageunits. A dose of a drug includes all or a portion of a therapeuticallyeffective amount of a particular drug that is to be administered in amanner and over a time sufficient to attain or maintain a desiredconcentration range of the drug, for instance, a desired concentrationrange of the drug in the immediate vicinity of a delivery needle in asolid tissue, and where the absolute amount of the drug that comprises adose will vary according to the drug, the subject, the solid tissue andother criteria with which the skilled practitioner will be familiar inview of the state of the medical and pharmaceutical and related arts. Incertain embodiments at least two doses of the drug may be administered,and in certain other embodiments 3, 4, 5, 6, 7, 8, 9, 10 or more dosesmay be administered.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,saline solution (e.g., normal saline, or isotonic, hypotonic orhypertonic sodium chloride), fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile. It may also bedesirable to include other components in the preparation, such asdelivery vehicles including but not limited to aluminum salts,water-in-oil emulsions, biodegradable oil vehicles, oil-in-wateremulsions, biodegradable microcapsules, hydrogels, and liposomes.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a conventional sustained drug release is also desired. Forparenteral administration, such as supplemental injection of drug, thecarrier preferably comprises water, saline, alcohol, a fat, a wax or abuffer biodegradable microspheres (e.g., polylactic galactide) may alsobe employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, itis preferable according to certain embodiments that the microsphere belarger than approximately 25 microns, while other embodiments are not solimited and contemplate other dimensions.

Pharmaceutical compositions may also contain diluents such as buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, an agent (e.g., a therapeutic drug ora candidate drug) is formulated as a lyophilizate using appropriateexcipient solutions (e.g., sucrose) as diluents.

For convenience, elements of various embodiments have been described ascomponents of a number of discrete assemblies. However, in practice,elements of some of the assemblies may be omitted or grouped with otherassemblies. Accordingly, the particular arrangements of embodimentsdisclosed above do not impose similar organization on other embodimentsor the claims.

When used to refer to agents delivered from needles, the term fluid isto be read broadly to read on any substance capable of flowing throughsuch a needle, including liquids, gases, colloids, suspended solids,etc.

The abstract of the present disclosure is provided as a brief outline ofsome of the principles of the invention according to one embodiment, andis not intended as a complete or definitive description of anyembodiment thereof, nor should it be relied upon to define terms used inthe specification or claims. The abstract does not limit the scope ofthe claims.

Elements of the various embodiments described above can be combined, andfurther modifications can be made, to provide further embodimentswithout deviating from the spirit and scope of the invention. All of theU.S. patents, U.S. patent application publications, U.S. patentapplications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide yet further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification, but should be construed toinclude all possible embodiments along with the full scope ofequivalents to which such claims are entitled. Accordingly, the claimsare not limited by the disclosure.

What is claimed is: 1.-103. (canceled)
 104. A method of evaluating oneor more candidate agents comprising: i) introducing said one or morecandidate agents to one or more positions in a tissue in vivo, and ii)evaluating an effect of said one or more candidate agents on saidtissue, wherein each of said one or more candidate agents is in apharmaceutical composition, and wherein at least one of said one or morepharmaceutical compositions comprises an indicator particle.
 105. Themethod of claim 104, wherein said tissue is a solid tumor.
 106. Themethod of claim 104, wherein said indicator particle is selected from ametallic particle, a fluorescent dye, a quantum dot, a quantum barcode,a radiographic contrast agent, and a magnetic resonance imaging contrastagent.
 107. The method of claim 106, wherein said indicator particle isa fluorescent dye.
 108. The method of claim 104, wherein at least one ofsaid candidate agents is a positive control or a negative control. 109.The method of claim 104, wherein said introducing in (i) is performedusing a device comprising one or more needles.
 110. The method of claim109, wherein said needles are arranged in an array.
 111. The method ofclaim 109, wherein said one or more needles are configured for passivedelivery of said one or more candidate agents.
 112. The method of claim109, wherein said device comprises needles with micropits over theirsurfaces.
 113. The method of claim 109, wherein said device comprisesneedles made from porous material.
 114. The method of claim 109, whereinat least one of said one or more needles is charged with a candidateagent by immersion in said candidate agent.
 115. The method of claim109, wherein each of said one or more needles is configured to deliversaid one or more candidate agents to spatially defined locations alongparallel axes in said solid tumor.
 116. The method of claim 109, whereineach of said one or more needles is configured to deliver said one ormore agents in a column-shaped region coaxial with respect to a deliveryaxis.
 117. The method of claim 109, wherein each of said one or moreneedles comprises a plurality of ports.
 118. The method of claim 117,wherein said plurality of ports are sized and positioned to deliver asubstantially equal amount of fluid.
 119. The method of claim 104,wherein said introducing in (i) comprises delivering less than 1 μL ofone or more candidate agents per needle to said solid tumor.
 120. Themethod of claim 109, wherein said one or more needles are close-ended.121. The method of claim 106, wherein said introducing in (i) comprisesdelivering, with said device, a plurality of different agents to aplurality of different positions within said solid tumor.
 122. Themethod of claim 104, wherein each of said one or more candidate agentsis introduced in an amount that is less than a minimal dose required toproduce a detectable effect in an adult or in an amount that isundetectable outside said solid tumor.
 123. The method of claim 104,further comprising introducing one or more indicators of efficacy intosaid solid tumor.
 124. The method of claim 104, wherein said evaluatingin (ii) comprises detecting a degree of permeation of said one or morecandidate agents through said solid tumor.
 125. The method of claim 104,wherein said evaluating in (ii) comprises assessing efficacy, activityor toxicity of said one or more candidate agents on said solid tumor.126. The method of claim 104, wherein at least one of said candidateagents is a chemotherapeutic.
 127. The method of claim 104, wherein atleast one of said candidate agents is a preclinical or clinical drug.